Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium.
We recently demonstrated that nitric oxide (NO) significantly contributes to the mitogenic effect of vascular endothelial growth factor (VEGF), suggesting a role for the NO pathway in the signaling cascade following kinase-derivative receptor activation in vascular endothelium. The aim of this study was to investigate the intracellular pathways linked to VEGF/NO-induced endothelial cell proliferation. We assessed the activity of the mitogen-activated protein kinase (MAPK) that is specifically activated by growth factors, extracellular-regulated kinase (ERK1/2), on cultured microvascular endothelium isolated from coronary postcapillary venules. ERK1/2 was immunoprecipitated, and its activity was assessed with an immunocomplex kinase assay. In endothelial cells exposed for 5 min to the NO donor drug sodium nitroprusside at a concentration of 100 microM, ERK1/2 activity significantly increased. VEGF produced a time- and concentration-dependent activation of ERK1/2. Maximal activity was obtained after 5 min of stimulation at a concentration of 10 ng/ml. The specific MAPK kinase inhibitor PD 98059 abolished ERK1/2 activation and endothelial cell proliferation in a concentration-dependent manner in response to VEGF and sodium nitroprusside. The NO synthase inhibitor Nomega-monomethyl-L-arginine, as well as the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked the activation of ERK1/2 induced by VEGF, suggesting that NO and cGMP contributed to the VEGF-dependent ERK1/2 activation. These results demonstrate for the first time that kinase-derivative receptor activation triggers the NO synthase/guanylate cyclase pathway to activate the MAPK cascade and substantiates the hypothesis that the activation of ERK1/2 is necessary for VEGF-induced endothelial cell proliferation.
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Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Morgagni 65, 50134 Florence, Italy.
Morbidelli
L
L
Cui
X L
XL
Douglas
J G
JG
Hood
J D
JD
Granger
H J
HJ
Ledda
F
F
Ziche
M
M
D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000254:growth & development; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D008222:Lymphokines / Q000494:pharmacology*; D020929:Mitogen-Activated Protein Kinase Kinases; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D009599:Nitroprusside / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011810:Quinoxalines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000502:physiology; D017978:Receptors, Growth Factor / Q000502:physiology; D040262:Receptors, Vascular Endothelial Growth Factor; D015398:Signal Transduction / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019323:omega-N-Methylarginine / Q000494:pharmacology
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D005419:Flavonoids; D008222:Lymphokines; D010069:Oxadiazoles; D047428:Protein Kinase Inhibitors; D011810:Quinoxalines; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D009599:Nitroprusside; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D006162:Guanylate Cyclase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium